The conversion of glucose to fructose by the enzyme xylose isomerase is an important industrial process because fructose is sweeter to human taste than an equivalent amount of glucose or sucrose. Fructose has nutritional advantages over glucose or sucrose as a sweetener because less fructose is needed to impart a desired level of sweetness, and because it does not support the growth of the bacteria responsible for dental plaque as well as does sucrose which is the only economically competitive sweetener. However, the maximum exploitation of these benefits depends on rendering fructose economically competitive with alternative sweeteners, by devising the least expensive process for manufacturing food-grade fructose.
Current industrial practice uses a single-step enzyme-catalyzed isomerization of glucose to an approximately equilibrated mixture of glucose and fructuse, known as high-fructose syrup. Using this process, at equilibrium, only approximately 50% of the glucose has been transformed (Tewari et al., Appld. Bioch. and Biotech. 11:17-24 (1985)). Because percentage conversion varies directly with temperature, the fructose yield, and potentially the process economics, benefit from performing industrial glucose isomerization at the highest practical temperature.
Enzymes which catalyze the isomerization of sugars, including glucose, have been isolated from various organisms, including Bacillus subtilis, Escherichia coli, Ampullariella species and several Streptomyces species. The Streptomyces enzyme commonly used for commercial fructose production is most accurately designated xylose isomerase (XI), because it has much higher activity in converting xylose to xylulose than turning glucose into fructose. For industrial use, the purified enzyme is immobilized by adsorption to a solid support packed into a column, or "reactor", through which a concentrated solution of glucose is passed at the highest feasible temperature. The enzyme near the reactor inlet experiences a high concentration of glucose and low concentration of fructose. The enzyme near the reactor outlet is exposed to approximately equal concentrations of glucose and fructose. At any level in the catalytic reactor, the isomerase catalytic rate (V) depends on glucose (S) and fructose (P) concentrations ([ ]) as indicated in the following rate equation: ##EQU1##
In this equation [E].sub.o is the total enzyme concentration, K.sub.S is the Michaelis constant for glucose, K.sub.P is the Michaelis constant for fructose, and V/[E].sub.o is the enzyme specific activity, an expression of the catalytic effectiveness per enzyme molecule. k.sub.cat.sbsb.f and k.sub.cat.sbsb.r report the intrinsic catalytic activities of the glucose-saturated and fructose-saturated enzyme active sites, respectively representing the maximum possible forward (glucose.fwdarw.fructose) and reverse (fructose.fwdarw.glucose) values of V/[E] for a given temperature and pH. K.sub.S, K.sub.p, k.sub.cat.sbsb.f, and k.sub.cat.sbsb.r, vary with temperature, generally increasing with increased temperature below the temperature range where conformational unfolding of the enzyme occurs. Although K.sub.S and K.sub.P do not necessarily equal the respective dissociation constants for glucose and fructose, they probably approximate the dissociation constants in the case of Streptomyces XI, and therefore are inversely related to the affinities of the enzyme for glucose and fructose substrates.
XI catalytic activity in the industrially relevant (forward) direction is enhanced by environmental or mutational changes which increase k.sub.cat.sbsb.f or K.sub.P or decrease k.sub.cat.sbsb.r or K.sub.S, increase the intrinsic forward catalytic efficiency or affinity for glucose or decrease the intrinsic reverse catalytic efficiency or affinity of XI for fructose. Currently used industrial glucose isomerization processes do not produce the maximum possible (equilibrium) percent conversion of glucose to fructose because the reaction slows as equilibrium is approached. Improvements which permit closer approach to equilibrium by weakening the fructose-XI interaction or by strengthening glucose-XI binding can be as valuable as improvements which permit conversion at higher temperature, where the equilibrium percent conversion is greater.
The preceding rate equation implies that there are many ways to change k.sub.cat.sbsb.f, k.sub.cat.sbsb.r, K.sub.S, or K.sub.P to get a net increase in V/[E].sub.o. Detrimental changes in one or more kinetic parameters can be outweighed by beneficial changes in others. Some combinations of changes would reduce net activity. Structural changes affecting activity will alter several or all of the parameters, not all of them favorably, for two reasons:
(a) The four kinetic parameters are inescapably linked through the Haldane relationship:
Keq=[P]equilibrium/[S]equilibrium=k.sub.cat.sbsb.f K.sub.p /k.sub.cat.sbsb.r K.sub.S
At a given temperature and pH, a change in one parameter must be accompanied by a balancing change in some combination of the others to preserve the value of K.sub.eq, the equilibrium constant; and
(b) The relatively few amino acid residues which line the xylose isomerase active site interact with glucose, fructose, and catalytic intermediates. These interactions determine the values of the four kinetic parameters. Changing any one active site residue will strengthen or weaken several of these interactions and therefore modify several parameters.
It is thus difficult to target a simple set of improvements in catalytic activity because a change which improves one parameter may have strongly damaging effects on others. However, atomic resolution i.e. x-ray crystallographic data on the xylose isomerase active site permits the selection of a limited number of protein structural changes to increase net catalytic activity, for example, by strengthening the binding of glucose or by weakening the binding of fructose.
Recently, computer-graphic examination of the active sites of enzymes other than XI has led to successful prediction of structural changes affecting k.sub.catr, K.sub.m, and substrate specificity for these enzymes (Wilkinson et al., Nature 307:187-188 (1984); and Craik et al., Science 228:291 (1985)).
In addition to identifying active site mutations that may improve kinetic parameters, computerized graphical examination of the atomic-resolution crystallographic data for XI also permits prediction of amino acid substitutions, insertions, or deletions to stabilize the enzyme toward conformational unfolding or inactivating chemical reactions. Following are several recent examples of structurally stablizing mutations accomplished by site-specific or random mutagenesis.
Replacement of a glycine residue located in an .alpha.-helix has conformationally stabilized a neutral proteinase, increasing the thermal melting temperature by several degrees centigrade (Imanaka et al., Nature 324:695 (1986)).
Replacement of amino acids in the hydrophobic core of a protein with aromatic residues such as tyrosine, especially at positions near preexisting clusters of aromatic side chains, has been shown to promote resistance to thermal inactivation in kanamycin nucleotidyl transferase (Liao et al., Biochem., 83:576-580 (1986)), and phage Lambda repressor (Hecht et al., Biochem., 81:5685-5689 (1984)).
The introduction of new disulfide bonds to create covalent crosslinks between different parts of a polypeptide has been used to improve the thermal stability of bacteriophage T4lysozyme (Perry et al., Science 226:555 (1984)), bacteriophage Lambda repressor (Sauer et al., Biochem., 125:5992 (1986)), E. coli dihydrofolate reductase (Villafranca et al., Biochem., 26:2182 (1987)), and subtilisin BPN' (Pantoliano et al., Biochem., 2077-2083 (1987)). A recently developed computer program (Pabo et al., Biochem., 25:5987-5991 (1986)) permits efficient scanning of the crystallographically determined three-dimensional structure of a protein to suggest those sites where insertion of two cysteines might lead to disulfide bonds which would not disrupt the larger-scale conformation while stabilizing the local conformation.
Deamidation of an asparagine residue near the inter-subunit interface of a homodimeric protein (triose phosphate isomerase) promotes irreversible thermal denaturation of this enzyme. Replacement of this asparagine with isoleucine enhanced thermal stability (Ahern et al., P.N.A.S. USA, 84:675-679 (1987)).
Fusion of the subunits of the homotetramaric enzyme, .beta.-galactosidase, by duplication and in-phase head-to-tail fusion of the structural gene for the enzyme, using a DNA polylinker coding for a number of additional amino acids, resulted in a protein that was more stable toward proteolysis and heat compared to the wild-type enzyme (Kuchinke et al., EMBO J., 4(4):1067-1073 (1985)).
Another class of potentially inactivating reactions include oxidation of amino acid residues at or near the active site of an enzyme, leading to a loss or reduction in catalytic activity. For example, oxidation of a key methionine residue in the protein subtilisin has been shown to lead to loss of proteolytic activity (Markland et al., in The Enzymes (P. Boyer, ed.) Vol. III:561 Academic Press (1971)). Replacement of that methionine by a serine, alanine or leucine residue resulted in an oxidation-resistant mutant protein (Estell et al., J. Biol. Chem. 260:6518-6521 (1985)).
Recent studies also have shown that some amino acid substitutions may have cumulative beneficial effects on thermalstability of the protein subtilisin (Bryan et al., J. Cellular Biochem Supc. 11C (N305) (1987); Matsumura et al., Nature (Letters) 323:356-358 (1986)).
Classical mutation of bacteria using radiation or chemicals has been used to produce mutant strains having different properties including altered protein activity. However, selective improvement of the organisms or the proteins has not been realized due to the randomness of the mutation process, which also requires tedious selection and screening steps to identify organisms which may possess the desired characteristics. Furthermore, with random mutagenesis an undesirable property may result along with the characteristic sought in the mutation.
More recently, random mutagenesis has been replaced by sitespecific (also known as primer-directed) mutagenesis. Sitespecific mutagenesis permits substitution, deletion or insertion of selected nucleotide bases within a DNA sequence encoding a protein of interest using synthetic DNA oligonucleotides having the desired sequence. Recombinant DNA procedures are used to substitute the synthetic sequence for the target sequence to introduce the desired mutation. (See Craik et al., Science, 228:291 (1985) for a review of these procedures). Development of the M13 bacteriophage vectors (Messing, in Methods in Enzymoloqy 101:20-78 (1983)) permits cloning of DNA fragments into singlestranded circular recombinants capable of autonomous replication. A modification of site-specific mutagenesis, termed gapped circle mutagenesis, provides an improved method for selective mutagenesis using oligonucleotide primers (Kramer et al., Nuc. Acids Res., 12:9441-9456 (1984)). Kits for carrying out sitespecific mutagenesis and the gapped circle method are commercially available.
Mutant xylose isomerases having characteristics which vary from native enzyme would be useful. In particular, mutant isomerases having enhanced oxidation and thermal stability would be useful to extend the commercial utility of the enzyme.
Unfortunately, unless proteins share regions of substantial sequence or structural homology, it is not possible to generalize among proteins to predict, based on beneficial mutation of one protein, precisely where the sequence encoding another protein should be changed to improve the performance of that protein. It is therefore generally necessary to undertake an analysis of the precise structural and functional features of the particular protein to be altered in order to determine which amino acids to alter to produce a desired result such as increased thermostability or catalytic activity.
The present invention provides mutated forms ("muteins") of enzymatically active procaryotic xylose isomerase. Analysis of the structure of Streptomyces rubiginosus xylose isomerase (XI) to select alterations encoding the enzyme to enhance stability and/or activity of the resulting XI muteins was undertaken. Site-specific mutagenesis of the sequence encoding the enzyme was designed to produce the muteins. Regions of structural homology between xylose isomerases from several microorganisms were identified.